TY  - JOUR
AU  - Wu, Guandi
AU  - Frankish, Jamie
AU  - Willemsen, Joschka
AU  - Ricken, Dominik
AU  - Becker, Jonas
AU  - Schweinoch, Darius
AU  - Beneke, Jürgen
AU  - Wüst, Sandra
AU  - Beil, Nina
AU  - Matula, Petr
AU  - Rohr, Karl
AU  - Erfle, Holger
AU  - Kaderali, Lars
AU  - Binder, Marco
TI  - High-throughput screening of E3 ubiquitin ligases identifies TRIM48 as a novel negative regulator of RIG-I signaling.
JO  - Cellular signalling
VL  - 134
SN  - 0898-6568
CY  - Amsterdam [u.a.]
PB  - Elsevier Science
M1  - DKFZ-2025-01414
SP  - 111973
PY  - 2025
N1  - #EA:D430#LA:D430#
AB  - The retinoic acid-inducible gene-I (RIG-I) signaling is crucial for cell-intrinsic innate antiviral immunity. Upon cytosolic detection of virus-associated RNA, it triggers a cascade inducing production of potent cytokines, mainly type I and III interferons (IFNs). While effective, dysregulated responses can harm the host, requiring tight pathway control. Here, we performed a comprehensive, systematic siRNA-based high-throughput screen across 616 established and putative E3 ubiquitin ligases for their impact on RIG-I signaling. We employed a fluorescence-based live-cell imaging assay in A549 cells to monitor nuclear translocation of IRF3 and NF-κB, two key transcription factors downstream of RIG-I. Candidate genes were validated in an orthogonal secondary screen, assessing their impact on the functional antiviral response to a Rift Valley Fever reporter virus. Fourteen hits showed consistent effects on RIG-I signaling across both screens. These genes were further validated and characterized by assessing IFN-β promoter reporter activity and IFNB1 mRNA levels upon dsRNA transfection. TRIM48 emerged as a highly robust negative regulator. Overexpression of TRIM48 suppressed RIG-I-mediated activation of IRF3 and NF-κB, reduced IFN and IFN-stimulated gene expression, and enhanced viral replication. Conversely, TRIM48 deficiency enhanced RIG-I signaling and inhibited viral replication. Notably, TRIM48 acts as an induced feedback regulator upon infection, and its effect depended on its enzymatic ubiquitin ligase activity. Our high-throughput screen provides an unbiased assessment of close to all E3 ubiquitin ligases for their regulatory effect in RIG-I signaling, and identified several interesting candidates for further investigation. TRIM48 was established as a negative feedback regulator of the RIG-I pathway.
KW  - E3 ubiquitin ligases (Other)
KW  - Innate antiviral immunity (Other)
KW  - RIG-I signaling (Other)
KW  - TRIM48 (Other)
KW  - siRNA screening (Other)
LB  - PUB:(DE-HGF)16
C6  - pmid:40609779
DO  - DOI:10.1016/j.cellsig.2025.111973
UR  - https://inrepo02.dkfz.de/record/302874
ER  -